RESUMEN
Within the genome, expressed genes marked by "open" chromatin are often adjacent to silent, heterochromatic regions. There are also regions containing neighboring active genes with different programs of expression. In both cases, DNA sequence elements may function as insulators, either providing barriers that prevent the incursion of heterochromatic signals into open domains or acting to block inappropriate contact between the enhancer of one gene and the promoter of another. The mechanisms associated with insulation are diverse: Enhancer-blocking insulation is largely associated with the ability to stabilize the formation of loop domains within the nucleus. Barrier insulation is often associated with the ability to block propagation of silencing histone modifications. Here, we provide examples of both kinds of insulator action, derived initially from studies of the compound insulator element at the 5' end of the chicken ß-globin locus. Such elements appear to have more general regulatory roles in the genome that have been exploited to provide insulator function where necessary to demarcate separate domains within the nucleus.
Asunto(s)
Cromatina/metabolismo , Elementos Aisladores/genética , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Factor de Unión a CCCTC , Pollos , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Elementos de Facilitación Genéticos/genética , Heterocromatina/metabolismo , Ácidos Hidroxámicos/farmacología , Modelos Biológicos , Unión Proteica/efectos de los fármacos , Proteínas Represoras/metabolismo , Ribonucleasa III/metabolismo , Globinas beta/genética , ADN Metiltransferasa 3BRESUMEN
Transferrin binding protein A (TbpA) is a TonB-dependent outer membrane protein expressed by pathogenic bacteria for iron acquisition from human transferrin. The N-terminal 160 residues (plug domain) of TbpA were overexpressed in both the periplasm and cytoplasm of Escherichia coli. We found this domain to be soluble and monodisperse in solution, exhibiting secondary structure elements found in plug domains of structurally characterized TonB-dependent transporters. Although the TbpA plug domain is apparently correctly folded, we were not able to observe an interaction with human transferrin by isothermal titration calorimetry or nitrocellulose binding assays. These experiments suggest that the plug domain may fold independently of the beta-barrel, but extracellular loops of the beta-barrel are required for ligand binding.
Asunto(s)
Neisseria meningitidis/química , Estructura Secundaria de Proteína , Proteína A de Unión a Transferrina/química , Animales , Dicroismo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Mutación , Estructura Terciaria de Proteína , Proteína A de Unión a Transferrina/genéticaAsunto(s)
Cromatina/química , Cromatina/genética , Animales , Factor de Unión a CCCTC , Pollos , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Impresión Genómica , Globinas/genética , Humanos , Elementos Aisladores , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismoRESUMEN
The mature human immunodeficiency virus type 1 protease rapidly folds into an enzymatically active stable dimer, exhibiting an intricate interplay between structure formation and dimerization. We now show by NMR and sedimentation equilibrium studies that a mutant protease containing the R87K substitution (PR(R87K)) within the highly conserved Gly(86)-Arg(87)-Asn(88) sequence forms a monomer with a fold similar to a single subunit of the dimer. However, binding of the inhibitor DMP323 to PR(R87K) produces a stable dimer complex. Based on the crystal structure and our NMR results, we postulate that loss of specific interactions involving the side chain of Arg(87) destabilizes PR(R87K) by perturbing the inner C-terminal beta-sheet (residues 96-99 from each monomer), a region that is sandwiched between the two beta-strands formed by the N-terminal residues (residues 1-4) in the mature protease. We systematically examined the folding, dimerization, and catalytic activities of mutant proteases comprising deletions of either one of the terminal regions (residues 1-4 or 96-99) or both. Although both N- and C-terminal beta-strands were found to contribute to dimer stability, our results indicate that the inner C-terminal strands are absolutely essential for dimer formation. Knowledge of the monomer fold and regions critical for dimerization may aid in the rational design of novel inhibitors of the protease to overcome the problem of drug resistance.
Asunto(s)
Proteasa del VIH/química , Proteasa del VIH/metabolismo , Pliegue de Proteína , Urea/análogos & derivados , Secuencia de Aminoácidos , Azepinas , Dimerización , Proteasa del VIH/genética , Inhibidores de la Proteasa del VIH/metabolismo , VIH-1/enzimología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Relación Estructura-Actividad , Urea/metabolismoRESUMEN
Here we describe the production of a rabbit polyclonal Ab (RAS1) raised against the stalk of murine CD23. RAS1 inhibits release of CD23 from the surface of both M12 and B cells resulting in an increase of CD23 on the cell surface. Despite this increase, these cells are unable to bind IgE as determined by FACS. CD23 has previously been shown to bind IgE with both a high (4-10 x 10(7) M(-1)) and low (4-10 x 10(6) M(-1)) affinity. Closer examination by direct binding of (125)I-IgE revealed that RAS1 blocks high affinity binding while having no effect on low affinity binding. These data support the model proposing that oligomers of CD23 mediate high affinity IgE binding. These experiments suggest that RAS1 binding to cell surface CD23 results in a shift from oligomers to monomers, which, according to the model, only bind IgE with low affinity. These experiments also suggest that high affinity binding of IgE is required for IgE regulation by CD23 and is demonstrated by the fact that treatment of Ag/Alum-immunized mice treated with RAS1 results in a significant increase in IgE production similar to the levels seen in CD23-deficient mice. These mice also had significantly decreased levels of serum soluble CD23 and Ag-specific IgG1. RAS1 had no effect on IgE or Ag-specific IgG1 production in CD23-deficient mice.
Asunto(s)
Inmunoglobulina E/biosíntesis , Receptores de IgE/fisiología , Animales , Anticuerpos/inmunología , Biopolímeros , Epítopos/inmunología , Citometría de Flujo , Regulación de la Expresión Génica/fisiología , Inmunoglobulina E/genética , Interleucina-4/farmacología , Interleucina-5/farmacología , Linfoma de Células B/patología , Sustancias Macromoleculares , Ratones , Ratones Endogámicos BALB C , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Conejos , Receptores de IgE/química , Proteínas Recombinantes/farmacología , Células Tumorales CultivadasRESUMEN
Engagement of Fc gamma receptors (Fc gamma Rs) with the Fc region of IgG elicits immune responses by leukocytes. The recent crystal structure of Fc gamma RIII in complex with IgG-Fc has provided details of molecular interactions between these components (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). One of the most intriguing issues is that glycosylation of IgG-Fc is essential for the recognition by Fc gamma Rs although the carbohydrate moieties are on the periphery of the Fc gamma RIII-Fc interface. To better understand the role of Fc glycosylation in Fc gamma R binding we prepared homogeneous glycoforms of IgG-Fc (Cri) and investigated the interactions with a soluble form of Fc gamma RIIb (sFc gamma RIIb). A 1:1 complex stoichiometry was observed in solution at 30 degrees C (K(d), 0.94 microm; Delta G, -8.4 kcal mol(-1); Delta H, -6.5 kcal mol(-1); T Delta S, 1.9 kcal mol(-1); Delta C(p), -160 cal mol(-1) K(-1)). Removal of terminal galactose residues did not alter the thermodynamic parameters significantly. Outer-arm GlcNAc residues contributed significantly to thermal stability of the C(H)2 domains but only slightly to sFc gamma RIIb binding. Truncation of 1,3- and 1,6-arm mannose residues generates a linear trisaccharide core structure and resulted in a significantly decreased affinity, a less exothermic Delta H, and a more negative Delta C(p) for sFc gamma RIIb binding, which may result from a conformational change coupled to complex formation. Deglycosylation of the C(H)2 domains abrogated sFc gamma RIIb binding and resulted in the lowest thermal stability accompanied with noncooperative unfolding. These results suggest that truncation of the oligosaccharides of IgG-Fc causes disorder and a closed disposition of the two C(H)2 domains, impairing sFc gamma RIIb binding.
Asunto(s)
Antígenos CD/metabolismo , Inmunoglobulina G/metabolismo , Oligosacáridos/metabolismo , Receptores de IgG/metabolismo , Rastreo Diferencial de Calorimetría , Inmunoglobulina G/química , Unión Proteica , Espectrometría de Masa por Ionización de Electrospray , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
The nuclear envelope proteins LAP2, emerin and MAN1 share a conserved approximately 40-residue 'LEM' motif. Loss of emerin causes Emery-Dreifuss muscular dystrophy. We have solved the solution NMR structure of the constant region of human LAP2 (residues 1-168). Human LAP2(1-168) has two structurally independent, non-interacting domains located at residues 1-50 ('LAP2-N') and residues 111-152 (LEM-domain), connected by an approximately 60-residue flexible linker. The two domains are structurally homologous, comprising a helical turn followed by two helices connected by an 11-12-residue loop. This motif is shared by subdomains of T4 endonuclease VII and transcription factor rho, despite negligible (< or =15%) sequence identity. NMR chemical shift mapping demonstrated that the LEM-domain binds BAF (barrier-to-autointegration factor), whereas LAP2-N binds DNA. Both binding surfaces comprise helix 1, the N-terminus of helix 2 and the inter-helical loop. Binding selectivity is determined by the nature of the surface residues in these binding sites, which are predominantly positively charged for LAP2-N and hydrophobic for the LEM-domain. Thus, LEM and LEM-like motifs form a common structure that evolution has customized for binding to BAF or DNA.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de la Membrana/química , Proteínas Nucleares , Secuencia de Aminoácidos , ADN/química , Humanos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Membrana Nuclear/metabolismo , Nucleoproteínas/metabolismo , Estructura Terciaria de Proteína , SolucionesRESUMEN
Serotonin N-acetyltransferase (AANAT) controls the daily rhythm in melatonin synthesis. When isolated from tissue, AANAT copurifies with isoforms epsilon and zeta of 14-3-3. We have determined the structure of AANAT bound to 14-3-3zeta, an association that is phosphorylation dependent. AANAT is bound in the central channel of the 14-3-3zeta dimer, and is held in place by extensive interactions both with the amphipathic phosphopeptide binding groove of 14-3-3zeta and with other parts of the central channel. Thermodynamic and activity measurements, together with crystallographic analysis, indicate that binding of AANAT by 14-3-3zeta modulates AANAT's activity and affinity for its substrates by stabilizing a region of AANAT involved in substrate binding.
Asunto(s)
Arilamina N-Acetiltransferasa/química , Estructura Cuaternaria de Proteína , Tirosina 3-Monooxigenasa/química , Proteínas 14-3-3 , Animales , Arilamina N-Acetiltransferasa/genética , Arilamina N-Acetiltransferasa/metabolismo , Calorimetría , Cristalografía por Rayos X , Genes Reporteros/genética , Humanos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ovinos , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
XRCC4 is essential for carrying out non-homologous DNA end joining (NHEJ) in all eukaryotes and, in particular, V(D)J recombination in vertebrates. Xrcc4 protein forms a complex with DNA ligase IV that rejoins two DNA ends in the last step of V(D)J recombination and NHEJ to repair double strand breaks. XRCC4-defective cells are extremely sensitive to ionizing radiation, and disruption of the XRCC4 gene results in embryonic lethality in mice. Here we report the crystal structure of a functional fragment of Xrcc4 at 2.7 A resolution. Xrcc4 protein forms a strikingly elongated dumb-bell-like tetramer. Each of the N-terminal globular head domains consists of a beta-sandwich and a potentially DNA-binding helix- turn-helix motif. The C-terminal stalk comprising a single alpha-helix >120 A in length is partly incorporated into a four-helix bundle in the Xrcc4 tetramer and partly involved in interacting with ligase IV. The Xrcc4 structure suggests a possible mode of coupling ligase IV association with DNA binding for effective ligation of DNA ends.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Sitios de Unión , Proteínas de Ciclo Celular/química , Cristalización , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , ADN Ligasa (ATP) , ADN Ligasas/química , ADN Ligasas/metabolismo , Proteínas de Unión al ADN/genética , Dimerización , Humanos , Sustancias Macromoleculares , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
Barrier-to-autointegration factor (BAF) is a highly conserved cellular protein that was identified by its activity in protecting retroviral DNA against autointegration. We show that BAF has the property of bridging double-stranded DNA in a highly ordered nucleoprotein complex. Whereas BAF protein alone is a dimer in solution, upon binding DNA, BAF forms a dodecamer with DNA bound at multiple discrete sites in the complex. The interactions between BAF and DNA are entirely nonspecific with respect to DNA sequence. The dual interaction of BAF with DNA and LAP2, a protein associated with the nuclear lamina, suggests a role for LAP2 in chromosome organization. Consistent with this idea, RNA interference experiments with Caenorhabditis elegans reveal a defect in mitosis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas Nucleares , Animales , Secuencia de Bases , Caenorhabditis elegans/metabolismo , Clonación Molecular , Cartilla de ADN , Ratones , Mitosis , Nucleoproteínas/metabolismo , Integración ViralRESUMEN
GATA-1 and other vertebrate GATA factors contain a DNA binding domain composed of two adjacent homologous zinc fingers. Whereas only the C-terminal finger of GATA-1 is capable of independent binding to the GATA recognition sequence, double GATA sites that require both fingers for high affinity interaction are found in several genes. We propose a mechanism whereby adjacent zinc fingers interact to influence the binding and transactivation properties of GATA-1 at a subset of DNA-binding sites. By using two such double GATA sites we demonstrate that the N-terminal finger and adjacent linker region can alter the binding specificity of the C-terminal finger sufficiently to prevent it from recognizing some consensus GATA sequences. Therefore, the two zinc fingers form a composite binding domain having a different DNA binding specificity from that shown by the constituent single C-terminal finger. Furthermore, we compare two of these double sites and show that high affinity binding of GATA-1 to a reporter gene does not necessarily induce transactivation, namely the sequence of the DNA-binding site can alter the ability of GATA-1 to stimulate transcription.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/química , ADN/metabolismo , Proteínas de Escherichia coli , Proteínas de Transporte de Monosacáridos , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras , Pollos , Clonación Molecular , Factores de Unión al ADN Específico de las Células Eritroides , Escherichia coli , Factor de Transcripción GATA1 , Humanos , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Vertebrados , Dedos de ZincRESUMEN
The DNA binding domain of GATA-1 consists of two adjacent homologous zinc fingers, of which only the C-terminal finger binds DNA independently. Solution structure studies have shown that the DNA is bent by about 15 degrees in the complex formed with the single C-terminal finger of GATA-1. The N-terminal finger stabilizes DNA binding at some sites. To determine whether it contributes to DNA bending, we have performed circular permutation DNA bending experiments with a variety of DNA-binding sites recognized by GATA-1. By using a series of full-length GATA-1, double zinc finger, and single C-terminal finger constructs, we show that GATA-1 bends DNA by about 24 degrees, irrespective of the DNA-binding site. We propose that the N- and C-terminal fingers of GATA-1 adopt different orientations when bound to different cognate DNA sites. Furthermore, we characterize circular permutation bending artifacts arising from the reduced gel mobility of the protein-DNA complexes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/química , Conformación de Ácido Nucleico , Plásmidos/química , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras , Pollos , ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Glutatión Transferasa , Humanos , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleótidos/química , Plásmidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Dedos de ZincRESUMEN
Immunoglobulin E (IgE) exhibits a uniquely high affinity for its receptor, FcepsilonRI, on the surface of mast cells and basophils. Previous work has implicated the third domain of the constant region of the epsilon-heavy chain (Cepsilon3) in binding to FcepsilonRI, but the smallest fragment of IgE that is known to bind with full affinity is a covalent dimer of the Cepsilon3 and Cepsilon4 domains. We have expressed the isolated Cepsilon3 in Escherichia coli, measured its affinity for FcepsilonRI, and examined its conformation alone and in the complex with FcepsilonRI. Sedimentation equilibrium in the analytical centrifuge reveals that this product is a monomer. The kinetics of binding to an immobilized fragment of the FcepsilonRI alpha-chain, measured by surface plasmon resonance, yields an affinity constant K(a) = 5 x 10(6) M(-)(1), as compared with 4 x 10(9) M(-)(1) for IgE. The circular dichroism spectrum and measurements of fluorescence as a function of the concentration of a denaturant do not reveal any recognizable secondary structure or hydrophobic core. On binding to the FcepsilonRI alpha-chain fragment, there is no change in the circular dichroism spectrum, indicating that the conformation of Cepsilon3 is unchanged in the complex. Thus the isolated Cepsilon3 domain is sufficient for binding to FcepsilonRI, but with lower affinity than IgE. This may be due to the loss of its native immunoglobulin domain structure or to the requirement for two Cepsilon3 domains to constitute the complete binding site for FcepsilonRI or to a combination of these factors.
Asunto(s)
Inmunoglobulina E/química , Receptores de IgE/metabolismo , Línea Celular , Dicroismo Circular , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Inmunoglobulina E/genética , Inmunoglobulina E/metabolismo , Cinética , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
We have investigated the partial specific volumes (2) (ml/g), hydration, and cosolvent interactions of rabbit muscle aldolase by equilibrium sedimentation in the analytical ultracentrifuge and by direct density increment (partial differential/partial differentialc(2))(mu) measurements over a range of sugar concentrations and temperature. In a series of sugars increasing in size, glucose, sucrose, raffinose, and alpha-cyclodextrin, (partial differential/ partial differentialc(2))(mu) decreases linearly with the solvent density rho(0). These sugar cosolvents do not interact with the protein; however, the interaction parameter B(1) (g water/g protein) mildly increases with increasing sugar size. The experimental B(1) values are smaller than values calculated by excluded volume (rolling ball) considerations. B(1) relates to hydration in this and in other instances studied. It decreases with increasing temperature, leading to an increase in (2) due to reduced water of hydration electrostriction. The density increments (partial differential/ partial differentialc(2))(mu), however, decrease in concave up form in the case of glycerol and in concave down form for trehalose, leading to more complex behavior in the case of carbohydrates playing a biological role as osmolytes and antifreeze agents. A critical discussion, based on the thermodynamics of multicomponent solutions, is presented.
Asunto(s)
Carbohidratos/química , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , alfa-Ciclodextrinas , Animales , Metabolismo de los Hidratos de Carbono , Ciclodextrinas/química , Glucosa/química , Glicerol , Cinética , Peso Molecular , Músculo Esquelético/enzimología , Conejos , Rafinosa/química , Solventes , Sacarosa/química , Termodinámica , Trehalosa , Ultracentrifugación/métodosRESUMEN
Antibodies are multifunctional molecules that following the formation of antibody antigen complexes, may activate mechanisms to effect the clearance and destruction of the antigen (pathogen). The IgG molecule is comprised of three globular protein moieties (2Fab+Fc) linked through a flexible hinge region. While the Fabs bind antigens, the Fc triggers effector mechanisms through interactions with specific ligands, e.g. cellular receptors (FcgammaR), and the C1 component of complement. Glycosylation of IgG-Fc has been shown to be essential for efficient activation of FcgammaR and C1. We report the generation of a series of truncated glycoforms of IgG-Fc, and the analysis of the contribution of the residual oligosaccharide to IgG-Fc function and thermal stability. Differential scanning microcalorimetry has been used to compare the stabilities of the homogeneous glycoforms of IgG1-Fc. The results show that all truncated oligosaccharides confer a degree of functional activity, and thermodynamic stability to the IgG1-Fc, in comparison with deglycosylated IgG1-Fc. The same truncated glycoforms of an intact IgG1 anti-MHC Class II antibody are shown to exhibit differential functional activity for FcgammaRI and C1 ligands, relative to deglycosylated IgG1. The minimal glycoform investigated had a trisaccharide attached to each heavy chain and can be expected to influence protein structure primarily in the proximity of the N-terminal region of the C(H)2 domain, implicated as a binding site for multiple effector ligands. These data provide a thermodynamic rationale for the modulation of antibody effector functions by different glycoforms.
Asunto(s)
Glicoproteínas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Oligosacáridos/inmunología , Receptores de IgG/inmunología , Rastreo Diferencial de Calorimetría , Glicosilación , Humanos , Desnaturalización Proteica , Transducción de Señal , Superóxidos/metabolismo , Temperatura , Células U937RESUMEN
Type II beta phosphatidylinositol phosphate kinase is a representative phosphatidylinositol phosphate kinase that is active against membrane-bound substrates. The structure of the enzyme contains a flattened basic face that spans the crystallographic dimer interface and is adjacent to the active site. Analytical ultracentrifugation shows that phosphatidylinositol phosphate kinase is a dimer in solution. Modeling suggested that the flattened face binds to acidic phospholipids by electrostatic interactions. The enzyme binds to acidic vesicles containing phosphatidylserine, phosphatidic acid, or phosphoinositides mixed with phosphatidylcholine, but not to neutral phosphatidylcholine vesicles. Binding to acidic vesicles is abolished in the presence of 1.0 M NaCl, consistent with an essential electrostatic contribution to the free energy of binding. The +14 charge on the flattened face of the dimer was reduced to +2 in the triple mutant Lys72Glu/Lys76Glu/Lys78Glu. The mutation has no effect on dimerization, but reduces the apparent KA for 25% phosphatidylserine/75% phosphatidylcholine mixed vesicles by 16-fold. The reduction in the level of binding can be ascribed to a loss of electrostatic interactions based on the finite difference solution to the Poisson-Boltzmann equation. The mutant reduces catalytic activity toward phosphatidylinositol 5-phosphate by approximately 50-fold. The wild-type enzyme binds half-maximally to phosphatidylinositol 4,5-bisphosphate-containing vesicles at a mole fraction of 0.3% in a phosphatidylcholine background, as compared to a 22% mole fraction in phosphatidylserine. The binding to phosphatidylinositol 4,5-bisphosphate-containing membranes is less sensitive to salt and to the triple mutation than binding to phosphatidylserine-containing membranes, suggesting that at least part of phosphatidylinositol 4,5-bisphosphate's interaction with the enzyme is independent of the flattened face. It is concluded that the flattened face of type II beta phosphatidylinositol phosphate kinase binds to membranes through nonspecific interactions, and that this interaction is essential for efficient catalysis.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/química , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fosfolípidos/metabolismo , 1-Fosfatidilinositol 4-Quinasa/genética , Sitios de Unión/genética , Activación Enzimática/genética , Ácido Glutámico/genética , Humanos , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos/química , Lisina/genética , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfolípidos/química , Unión Proteica/genética , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Electricidad Estática , UltracentrifugaciónRESUMEN
Glycosylation of the Fc region of IgG (IgG-Fc) is essential for the full expression of Fc effector functions. The profound differences in functional activity observed between glycosylated and aglycosylated IgG have not previously been paralleled by the demonstration of large-scale structural changes. In the present study differential scanning microcalorimetry (DSMC) was used to investigate IgG-Fc glycoprotein stability and to determine the thermodynamic parameters for thermal unfolding, which will include a contribution from the intra-molecular oligosaccharide-protein interactions. The thermogram obtained for glycosylated IgG1-Fc yielded two clearly defined transitions whilst the glycosylated IgG4-Fc exhibited a single transition. The methodology was also able to reveal measurable differences in the stability of IgG4-Fc glycoforms differing by the presence or absence of terminal galactose residues; deglycosylated IgG4-Fc exhibited two transitions with evidence for destabilisation of the C(H)2 domain.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Rastreo Diferencial de Calorimetría , Glicosilación , Calor , Humanos , Conformación Proteica , Desnaturalización ProteicaRESUMEN
The solution structure of the human barrier-to-autointegration factor, BAF, a 21,000 Mr dimer, has been solved by NMR, including extensive use of dipolar couplings which provide a priori long range structural information. BAF is a highly evolutionarily conserved DNA binding protein that is responsible for inhibiting autointegration of retroviral DNA, thereby promoting integration of retroviral DNA into the host chromosome. BAF is largely helical, and each subunit is composed of five helices. The dimer is elongated in shape and the dimer interface comprises principally hydrophobic contacts supplemented by a single salt bridge. Despite the absence of any sequence similarity to any other known protein family, the topology of helices 3-5 is similar to that of a number of DNA binding proteins, with helices 4 and 5 constituting a helix-turn-helix motif. A model for the interaction of BAF with DNA that is consistent with structural and mutagenesis data is proposed.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Nucleares , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Cristalografía por Rayos X , ADN/química , ADN Viral/genética , Dimerización , Secuencias Hélice-Giro-Hélice , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Retroviridae/fisiología , Homología de Secuencia de Aminoácido , Integración Viral/fisiologíaRESUMEN
We have employed isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy to characterize the binding of soluble fragments of IgE (IgE-Fc and Fc epsilon 3-4) to a soluble fragment of the high-affinity receptor Fc epsilon RI alpha-chain (sFc epsilon RI alpha). The thermodynamic parameters for the interaction of IgE-Fc and Fc epsilon 3-4 with sFc epsilon RI alpha, determined using ITC, confirm the earlier conclusion that the C epsilon 2 domain is not involved in the interaction and that the stoichiometry of both complexes is 1:1. For both IgE-Fc and Fc epsilon 3-4, the value of Delta H degrees is -36.9 +/- 4.6 kcal mol-1 at 37.3 degreesC and Delta Cp degrees is -820 +/- 120 cal mol-1 K-1. The temperature at which DeltaS degrees is zero is 284 +/- 1 K, indicating that the entropy contribution to the thermodynamics of association is unfavorable at physiological temperature. Of particular interest is the large value of Delta Cp degrees. The large surface area of IgE and Fc epsilon RI alpha that is implicated in complex formation from previous mutagenesis studies on the two proteins may account in part for the magnitude of Delta Cp degrees. Additional contributions may arise from hydration within the binding site and changes in tertiary structure of the individual components of the complex. However, the CD spectra of IgE, IgE-Fc, and Fc epsilon 3-4 complexes with sFc epsilon RI alpha are merely the sum of the spectra of their individual components, indicating that the secondary structure of the immunoglobulin domain folds are preserved on complex formation. Thus, any change in tertiary structure must be limited to the relative disposition of the immunoglobulin domains C epsilon 3 and C epsilon 4 in IgE and the two immunoglobulin-like domains in the alpha-chain of Fc epsilon RI.